The goal of this proposal is to define the mechanism by which GTP participates in mitochondrial protein import using yeast as a model system. We have demonstrated for first time that GTP hydrolysis plays a direct and essential role during import of urea-denatured precursors into the matrix. Import of these denatured precursors does not depend on ATP-dependent interactions with cytosolic chaperones. Both external GTP and matrix ATP are required for efficient import. Protein import into or across mitochondrial outer and inner membranes is a multi-step process. GTP may regulate recognition and subsequent unfolding of precursors at the mitochondrial surface and/or provide energy for transmembrane movement of precursors into the organelle. The first objective is to define the exact steps that require GTP. The GTP effect is likely mediated by GTP-binding proteins. Thus far, a GTP-binding protein has not been identified as part of the outer (Tom) or inner membrane (Tim) translocases. The second objective is to identify these critical GTP-binding proteins. A chimeric protein (pPHPrA) has been constructed, comprising an authentic N-terminal mitochondrial precursor (delta-1 pyrroline-5-carboxylate dehydrogenase) lined, through glutathione S-transferase, to IgG binding domains derived from staphylococcal Protein A. The native pPGPrA becomes irreversibly arrested, both in vitro and in vivo, while being translocated across the outer and inner membranes of mitochondria. During in vivo import of pPGPrA, the outer and inner membranes of mitochondria become progressively "zippered" together, forming long stretches of close contact. Affinity chromatography of solubilized ~zippered~ membranes has identified several core components of both translocation channels. These proteins are specifically associated with the arrested PGPrA intermediates. They include three GTP- binding proteins, as well as all know essential elements of Tom and Tim. The final goal is to determine how the newly identified GTP- binding proteins participate (together with other Toms and Tims) in distinct stages during mitochondrial protein import. This project is of direct health-related significance, since perturbation of mitochondrial function is associated with specific mitochondrial disease, aging, and certain neurodegenerative disorders. There is strong evidence that the constituents of mitochondrial translocases and their role in protein import are conserved between yeast and humans.